Extensive research has been devoted to exploring the therapeutic potential of nucleic acids, including small interfering (siRNA), microRNA (miRNA), catalytic RNA (ribozymes), aptamer oligonucleotides (oligonucleotides with exquisite roles similar to protein receptors), and antisense oligonucleotides. Theoretically, when designed appropriately, nucleic acids delivered into biological systems will participate in cellular activities, such as RNA interference or gene silencing, to abolish specific gene expression in cells and to attain more precise therapeutic targeting than typical small molecule drugs. Nucleic acid-based therapeutics have shown promise for treating a variety of human genetic diseases and microbial infections. Recent progress has resulted in some antisense oligonucleotides and aptamer RNA reaching clinical applications, while a significant number of clinical trials for siRNA are underway.
The direct use of nucleic acids for treating diseases, however, faces serious hurdles. Difficulties include cell specificity, inefficient cellular uptake of nucleic acids, and inaccessibility of nucleic acids to cell nuclei, due primarily to ineffective translocation of nucleic acids across biological barriers after administration. Consequently, successful use of nucleic acids in clinical practice will not be achieved until there are better strategies for targeted and efficient delivery of nucleic acids to cells and tissues. The critical issue of efficient target delivery for nucleic acids has been studied by many laboratories through chemical modification of nucleic acids to improve stability and cellular delivery properties of nucleic acids in vivo.
Developed methods include conjugating cellular surface receptor-specific ligands with various nucleic acid-containing nanocarriers or covalently linking cellular surface receptor-specific ligands with nucleic acids directly.
The use of peptides as ligands to traffic nucleic acids across the plasma membrane has been extensively investigated in the development of effective nucleic acid-based therapeutic agents. Conjugating nucleic acids such as oligonucleotides with cell-penetrating peptides (CPPs) or cell-targeting peptides (CTPs) to acquire peptide-oligonucleotide conjugates (POCs) has created appropriate designs to circumvent cellular delivery or cell specificity problems inherited from administrating only oligonucleotides in clinics. CPPs, including the Tat, the Antennapedia, the CyLoP-1 (a cysteine-rich CPP), and the (KFF)3K peptides, are either protein-derived or artificially developed short sequences (10-16 amino acids), and they are able to spontaneously cross cellular barriers when provided in extracellular media. The unique cell permeability properties of CPPs significantly improve the uptake efficiency of oligonucleotides in POCs by cells and facilitate broader uses of POCs in science and medicine.
POCs are primarily prepared by coupling peptides with oligonucleotides after solid-phase synthesis (fragment coupling strategy) or directly synthesized through stepwise solid-phase reactions (online solid-phase synthesis). To achieve peptide-oligonucleotide conjugations, current POC synthesis methods typically require previous incorporations of additional functionalities in peptides, oligonucleotides, or both. The requirement to have additional functional groups renders these methods inefficient, inconvenient, and not cost-effective for academia or industry. The development of a facile approach to exploit readily available functionalities, such as hydroxyl or phosphate groups, in standard oligonucleotides in order to synthesize POCs with high purity and yields is crucial to the advancement of POC applications.
Regioselective modifications of biomolecules with tags, probes or other biological molecules have been a critical tool which significantly advanced biomolecular studies for fundamental research and clinical application. In nucleic acids, site-specific modifications of smaller DNA/RNA such as oligonucleotides can be achieved through phosphoramidite chemistry to link predefined chemical moieties to positions in specific nucleotides during solid-phase oligonucleotide synthesis. However, the solid-phase chemistry approach for regioselective modifications of oligonucleotides suffers from inherited drawbacks including limits on the length of synthesized oligonucleotides and on the variety of their incorporated chemical functionality.
To complement the shortcomings of solid-phase oligonucleotide synthesis, many site-specific post-synthetic modification methods for nucleic acids have been studied and adapted to any size of nucleic acid and a broad diversity of chemical groups integrated into the nucleic acids. Nevertheless, recently developed post-synthetic modification methods for nucleic acids rely on enzyme catalysis to carry out chemical transformations but are unable to provide a universal strategy for both DNA and RNA modifications. Moreover, the required expensive enzymes and specific substrates in enzymatic reactions further stymie the efforts to modify nucleic acids with various chemical entities within reasonable costs.